ABSTRACT Most solid tumors share two hallmarks that could be targets for cancer therapy: Chromosomal instability (CIN) and aneuploidy, with tumor cells generally carrying far more chromosomes than their euploid counterparts. CIN is defined by elevated frequencies of whole-chromosome missegregation, which in turn can lead to aneuploidy, an abnormal number of chromosomes. The conserved kinase Mps1, which is one of the most commonly over-expressed proteins in tumors with aneuploidy/CIN, is a promising target for cancer therapy. This kinase functions mainly to ensure successful chromosome segregation by promoting the correct attachment of chromosomes to microtubules. Interestingly, abnormally high levels of Mps1 are found in tumors with elevated chromosome numbers, and Mps1 is protective for these cells, making these tumor cells much more vulnerable to reductions in Mps1 activity than diploid cells. The recent development of selective and orally bioavailable small-molecule inhibitors of Mps1 has reinforced its potential as a therapeutic target for cancer. The major limitation of the current anti-Mps1 compounds, which induce its full inactivation, is the fact that those treatments can also have toxic impact on the physiology of normal cells. Mps1 phosphorylates many target proteins that are involved in several essential cellular processes. Critically, it is not known which of these targets and processes are the ones that are protective for aneuploid tumor cells. In this proposal, we will aim to identify the Mps1-dependent process and specific proteins that protect aneuploid tumor cells, using yeast cells with elevated chromosome number as a model system. As has been seen in human cells, yeast polyploid cells are exquisitely sensitive to reductions in Mps1 activity. We will interrogate the yeast gene set to identify genes whose levels are critical for the Mps1-target interaction that is critical for polyploid cells. The analysis of this interactome will identify direct Mps1 target proteins that are necessary for the protective function. In parallel, we will dissect the domains of Mps1 critical for its protective function. Combined with knowledge of putative targets, this information will provide critical tools for the analysis of the Mps1-target interaction. Successful completion of these experiments would provide fundamental information about how Mps1 interacts with its target proteins and will have important implications for the design of therapeutics that specifically target Mps1-target interactions that are protective for tumors.